Renal hypertrophy is among the earliest responses to diabetes and predicts the development of microalbuminuria. One critical area is how kidney cells accumulate the protein needed for growth. Diabetic renal hypertrophy involves both stimulation in protein synthesis and blockade of protein degradation (PD). In diabetes, our work has shown that the kidney suppresses PD through a lysosomal system leading to the accumulation of proteins that are substrates for destruction in lysosomes by chaperone-mediated autophagy (CMA). CMA destroys particular proteins important for renal tubular cell growth. However, CMA also destroys proteins that have undergone oxidative modification. In vitro, epidermal growth factor (EGF) signaling reduces CMA, while H2O2 stimulates it. However, both EGF and H2O2 activate signaling through the Akt protein kinase [unreadable] which reduces CMA in vivo. These conflicts between oxidative and growth factor effects on CMA raise some fundamental questions. 1) What mechanism explains two stimuli that act oppositely on CMA acting the same way on the signaling that regulates CMA? 2) How do oxidation and growth factors interact in controlling CMA in diabetic hypertrophy? 3) What is the consequence of protein oxidation on suppressing CMA when there is oxidative stress? We hypothesize that growth factors and oxidative stress have antagonistic effects on CMA in part through interactions on a down stream mediator of Akt signaling the class O F box transcription factors (FoxOs). Our Aims under this hypothesis are 1) To examine FoxO signaling in response to H2O2 and EGF and 2) To examine the interaction between H2O2 and EGF signaling on CMA and growth. Our second hypothesis is that oxidized proteins accumulate as CMA is suppressed during hypertrophy. Our Aims under this hypothesis are 3) To examine how altering CMA affects growth and accumulation of oxidized proteins in cell culture and 4) To examine proteolysis and protein oxidation in diabetic renal cortex when CMA is altered by dietary manipulation. To accomplish these objectives, we will use adenoviral expression vectors and siRNA [unreadable] techniques to manipulate molecules in the FoxO signaling pathway and components of the CMA proteolytic system. We will measure FoxO signaling, protein turnover, cell growth, reactive oxygen species production and the accumulation of oxidized proteins. Finally, we will test whether modified diets that reduce renal hypertrophy and diabetic kidney damage activate CMA and reduce accumulation of oxidized proteins. PUBLIC HEALTH RELEVANCE Our laboratory has observed that when kidneys grow in diabetes (hypertrophy), they decrease the breakdown of certain proteins important for growth. Certain signaling molecules in the diabetic kidney reduce the amount of protein destroyed in a process called chaperone-mediated autophagy. This proposal studies how this same process may be important for destroying proteins damaged by diabetes that contribute to kidney disease. [unreadable] [unreadable] [unreadable]